Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy

Fleissner, Mark R. and Bridges, Daniel M. and Brooks, Evan K. and Cascio, Duilio and Kálai, Tamás and Hideg, Kálmán and Hubbell, Wayne L. (2010) Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy. Proceedings of the National Academy of Sciences, 108. pp. 16241-16246. ISSN 0027-8424

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A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i þ 3 or i and i þ 4 in an α-helix, at i and i þ 2 in a β-strand, or with cysteine residues in adjacent strands in a β-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.

Item Type: Article
Subjects: Q Science / természettudomány > Q1 Science (General) / természettudomány általában
Depositing User: Dr. Tamás Kálai
Date Deposited: 15 Jan 2015 11:39
Last Modified: 15 Jan 2015 11:39

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